1. Field of the Invention
This invention relates to proteins. More particularly, it relates to proteins modified by attachment to organic polymers. Even more particularly, this invention relates to: (a) such a protein which has been modified by contacting it with an isocyanate-capped liquid polyurethane prepolymer to form a reaction product which is soluble or dispersible in water. Still more particularly, this invention relates to such protein and such process where said protein is an enzyme, an antibody, or an antigen and the polymer is a poly(urea-urethane).
The aqueous dispersions of protein/polymer reaction product are formed by dissolving the protein in an isocyanate-capped liquid polyurethane prepolymer and dispersing the resulting solution in water.
2. Description of the Prior Art
A review of enzyme technology was published in the Aug. 18, 1975 issue of Chemical & Engineering News (pp. 22-41).
The term "soluble bound enzymes" is employed in the prior art to designate the reaction product of a protein with a polymeric material (natural or synthetic). The reaction product is soluble or dispersible in liquid media, usually water. The protein/polymer bond is generally covalent although adsorption techniques have also been employed. The soluble bound proteins frequently exhibit biological reactivity similar in nature to that of the free protein. Some of the soluble bound proteins have exhibited increased solubility, and/or decreased antigenicity and other improvements in comparison with the free unmodified proteins.
In a published thesis (M. G. Brattain, Rutgers University, 1974 -- Modification of L-Asparaginase by Water Soluble Polymers) bonding via cyanuric chloride of asparaginase to polyethylene glycols and other polymers is described. The bound products are water-dispersibleand exhibit some improvement in stability as well as decreased antigenicity. The present invention is similarly concerned with binding enzymes to polyethylene glycols although the method of bonding and method of production differs greatly from the teachings of the Brattain thesis.
Additionally U.S. Pat. No. 3,574,062 (195/63, Sato) teaches a method for preparing a bound protein (an enzyme) wherein a polyester polyurethane is diazotized with a diazonium salt of an amino acid and then coupled with a nonenzymatic animal protein to form a diazotized polyurethane which is reacted with an enzyme to form the immobilized enzyme.
U.S. Pat. No. 3,705,084 (195/63, Reynolds) teaches a flow-through enzyme reactor comprising: (a) a macroporous reactor core; (b) a polymeric surface (which can be a polyurethane resin) on the reactor core; (c) an enzyme adsorbed on the polymeric surface and crosslinked in place thereon by a difunctional agent (e.g. a polyisocyanate).
Reynolds prepares the immobilized enzyme for his reactor by adsorbing an active enzyme on a polymeric surface and further immobilizing the enzyme by crosslinking it is place with a crosslinking agent such as a monomeric polyisocyanate.
German Offenlegungsschrift No. 2,319,706 published Nov. 15, 1973 teaches an enzyme bound to a polyurethane foam and a method for preparing such bound enzyme.
U.S. Pat. No. 3,791,927 (195/68, forgione et al) teaches a water-insoluble bound protein (enzyme) entrapped within the cells of a self-supporting reticulated cellular material (which can be a polyurethane foam), the protein (enzyme) being bound to the cellular material.
U.S. Pat. No. 3,672,955 (195/68, Stanley) teaches a process for preparing a bound protein (enzyme) comprising: (a) emulsifying an aqueous dispersion of the enzyme with a solution of a polyisocyanate in a volatile water-immiscible solvent (e.g., methylchloroform); (b) admixing the resulting emulsion with a solid particulate carrier; and (c) evaporating the solvent therefrom. Stanley's polyisocyanate can be an isocyanate-capped liquid polyurethane prepolymer. Said U.S. Pat. No. 3,672,955, in its entirety, is incorporated herein by reference.
It is noted that, in his Example 3, Stanley reports the binding of an enzyme component (a peroxidase) of a fermentation broth by admixing a portion of the broth with a polyisocyanate dissolved in methylchloroform. It seems probable that, under Stanley's reaction conditions, any other enzymes which were present in the broth would have been immobilized (rendered insoluble in water, i.e., bound).
Silman et al, Annual Review of Biochemistry, 1966, 35 (Part 2), pages 873-908 presents a review of methods for preparing water-insoluble derivatives of enzymes, antigens, and antibodies.
Singer, Nature, 1959, 183, 1523-1524 teaches a method for reacting a protein with a diisocyanate (m-xylene diisocyanate).
U.S. Patent Application Ser. No. 250,012, filed May 3, 1972, and now abandoned (Wood et al, inventors) which is assigned to W. R. Grace & Co. teaches, in Example 21, a foamed polyurethane comprising an immobilized enzyme (urease), a method for preparing such immobilized enzyme, and a method for using it.
Said Application Ser. No. 250,012 also teaches, e.g. in Claim 8, a foamable composition comprising: (a) an isocyanate-capped polyurethane prepolymer; (b) water; and (c) biostats, fungicides, or enzymes. A similar teaching occurs in claim 7 of the above-mentioned German Offenlegungsschrift No. 2,319,706.
U.S. Pat. No. 3,929,574, Wood et al, teaches the preparation of a bound (immobilized) protein, an enzyme, by a process comprising contacting an isocyanate-capped liquid polyurethane prepolymer with an aqueous dispersion of the enzyme under foam-forming conditions, whereby the polyurethane foams and the enzyme become integrally bound to the resulting polyurethane foam.
It is noted that, in said U.S. Pat. No. 3,929,574, Wood et al reports, in Example 1, that an enzyme (cellulase) present in a fermentation broth was immobilized (bound or rendered insoluble) by admixing the broth with an isocyanate-capped liquid polyurethane prepolymer under conditions which produced a foam. It seems probable that, under the conditions of said Example 1, any other enzymes present in the broth would have been immobilized.
U.S. Pat. No. 3,905,923 (260/2.5 AD, Klug) teaches an immobilized enzyme system formed from an enzyme and a hydrophilic poly(urea-urethane) foam, the foam surrounding, entrapping, and supporting the enzyme in an active configuration. The hydrophilic foam is formed by the reaction of water with a hydrophilic isocyanate-capped polyoxyalkylene prepolymer.
Isocyanate-capped polyurethane prepolymers are well known to those skilled in the art. See for example: (a) the penultimate paragraph on page 854 of Volume 9 of the Second Edition of the Kirk-Othmer "Encyclopedia of Chemical Technology", John Wiley and Sons, Inc., New York, N. Y., or (b) the third full paragraph in the left-hand (first) column of page 872 of the Second Edition of "The Encyclopedia of Chemistry", George L. Clark, Editor, Reinhold Publishing Corporation, New York, N. Y.
T. Richard and N. F. Olson, "Immobilized Enzymes in Food and Microbial Processes", Plenum Press, New York, N. Y., 1974, pages 35-36 teach the formation of a bound (immobilized) enzyme by reacting the enzyme, water, and a polyisocyanate polymer.
Weetall, Journal of Bacteriology, volume 93, pages 1876-1880 (1967) teaches the isolation and purification of large quantities of bacterial specific antibodies by using polymerized microorganisms as a specific immunoadsorbent. The microorganisms were polymerized by reaction with tetrazotized benzidine.